Progenitor cell differentiation in general and especially of neural progenitor cells (NPC) is not yet clearly understood. One mechanism of differentiation control is based on the activation of TCF-dependent transcription by ß-catenin, a key player in the ß-catenin mediated Wnt-pathway, whereby gene activation depends on ß-catenin translocation into the nucleus. We quantified ß-catenin signals in different cellular compartments during the early differentiation phase of the cells by semi-automatic quantitative analysis of confocal image stacks using Imaris™ (Bitplane). To investigate the influence of Wnt signals on the ß-catenin localization, the NPC, derived from human embryonic midbrain, were treated with either the glycogen synthase kinase-(GSK)-3ß inhibitor SB216763, or Wnt-inhibitory-factor-I (WIF-I), an inhibitor of free Wnt proteins. Wnt3a, a stimulator of the canonical Wnt-pathway, and Dickkopf-1 (Dkk1), the respective inhibitor, was used to investigate the upstream effects of this pathway. The results show that the nuclear ß-catenin concentration increases after induction of cell differentiation. Treatment with SB216763 and Wnt-3a enhanced this effect, whereas WIF-I and Dkk1 treatment suppressed it. The development of neuronal phenotypes was quantified in parallel to support the evidence that the differentiation is controlled by the Wnt-pathway. This precise quantification of fluorescence signals in 3D volumes correlates with neuronal phenotype development and therefore provides a means to investigate protein translocation processes between cellular compartments. Here we also show the comparison of different phenotypes - e.g. neurons and glia. Therefore this approach is suitable to compare the differentiation of certain cell types in a heterogeneous population.

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